key: cord-0880206-znut8otv
authors: VanBlargan, Laura A.; Adams, Lucas J.; Liu, Zhuoming; Chen, Rita E.; Gilchuk, Pavlo; Raju, Saravanan; Smith, Brittany K.; Zhao, Haiyan; Case, James Brett; Winkler, Emma S.; Whitener, Bradley M.; Droit, Lindsay; Aziati, Ishmael D.; Bricker, Traci L.; Joshi, Astha; Shi, Pei-Yong; Creanga, Adrian; Pegu, Amarendra; Handley, Scott A.; Wang, David; Boon, Adrianus C.M.; Crowe, James E.; Whelan, Sean P.J.; Fremont, Daved H.; Diamond, Michael S.
title: A potently neutralizing SARS-CoV-2 antibody inhibits variants of concern by utilizing unique binding residues in a highly conserved epitope
date: 2021-08-19
journal: Immunity
DOI: 10.1016/j.immuni.2021.08.016
sha: 83b45092c152638cf306a5a97633345e1d8a99e4
doc_id: 880206
cord_uid: znut8otv

With the emergence of SARS-CoV-2 variants with increased transmissibility and potential resistance, antibodies and vaccines with broadly inhibitory activity are needed. Here we developed a panel of neutralizing anti-SARS-CoV-2 monoclonal antibodies (mAbs) that bound the receptor binding domain of the spike protein at distinct epitopes and blocked virus attachment to its host receptor, human angiotensin converting enzyme-2 (hACE2). Although several potently neutralizing mAbs protected K18-hACE2 transgenic mice against infection caused by ancestral SARS-CoV-2 strains, others induced escape variants in vivo or lost neutralizing activity against emerging strains. One mAb, SARS2-38, potently neutralized all SARS-CoV-2 variants of concern tested and protected mice against challenge by multiple SARS-CoV-2 strains. Structural analysis showed that SARS2-38 engaged a conserved epitope proximal to the receptor binding motif. Thus, treatment with or induction of neutralizing antibodies that bind conserved spike epitopes may limit the loss of potency of therapies or vaccines against emerging SARS-CoV-2 variants.

(Iota), and B.1.617.1 (Kappa)) encoding these mutations. In contrast, SARS2-38 binds an epitope 80 centered on residues K444 and G446 and neutralized all tested VOCs and VOIs. Analysis of a 81 cryo-electron microscopy (cryo-EM) structure of SARS2-38 bound to spike reveals that this 82 mAb binds a conserved epitope on the RBD that is also engaged, albeit through distinct 83 geometries, by other neutralizing and protective human mAbs. Thus, treatment with mAbs or 84 induction of pAbs targeting this conserved region of the RBD may confer protection against 85 many emerging SARS-CoV-2 variants. 86

Development and characterization of anti-SARS-CoV-2 mAbs. We generated a panel 88 of anti-SARS-CoV-2 mAbs from BALB/c mice that were immunized with purified RBD and/or 89 ectodomain of the spike protein mixed with AddaVax™, a squalene-based adjuvant (Fig 1) . 90

After splenocyte-myeloma fusions, hybridoma supernatants were screened for antibody binding 91 to recombinant spike protein and permeabilized SARS-CoV-2-infected Vero cells by ELISA and 92 flow cytometry, respectively. Sixty-four hybridomas producing anti-SARS-CoV-2 antibodies 93 were cloned by limiting dilution. Forty-three of these mAbs bound to recombinant RBD and 94

were selected for further study because prior experiments showed this class included potently 95 inhibitory antibodies (Barnes et al., 2020; Baum et al., 2020a; Cao et al., 2020; Tortorici et al., 96 2020; Zost et al., 2020) ; the majority of these mAbs were of the IgG1 subclass (Fig 1) . 97

The mAbs were evaluated by competition binding analysis using three previously 98 characterized human mAbs that recognize distinct antigenic sites on the RBD (COV2-2196, 99 COV2-2130, and CR3022) (Yuan et al., 2020; Zost et al., 2020) (Fig 1) . Although the relatively 100 large size of antibodies limits the precision of this mapping approach, competition binding 101 analysis can allow classification of mAb interaction regions in a high-throughput manner. Eight 102 mAbs competed for spike protein binding with the neutralizing mAb COV2-2196 only, eight 103 mAbs competed with the neutralizing mAb COV2-2130 only, four mAbs competed with both 104 COV2-2196 and COV2-2130, and twenty mAbs competed with CR3022, a mAb that recognizes 105 a more conserved, cryptic epitope on the SARS-CoV-2 spike protein distal from the receptor 106 binding site. Three RBD-binding mAbs did not compete with COV2-2196, COV2-2130, or 107 CR3022. Based on the binding analysis, mAbs were divided into five competition groups, A-E 108 (Fig 1) . 109 J o u r n a l P r e -p r o o f One potential mechanism of antibody-mediated neutralization of SARS-CoV-2 is through 110 inhibition of viral spike protein binding to the human ACE2 receptor. The COV2-2196 epitope 111 directly overlaps the ACE2 binding site on RBD, whereas the COV2-2130 epitope lies proximal 112 to residues in the RBM that interact with ACE2 (Dong et al., 2021) ; nonetheless, both mAbs can 113 block spike binding to ACE2. In contrast, CR3022 engages the base of the RBD and does not 114 block ACE2 binding to spike (Yuan et al., 2020) . Of the 43 RBD-binding antibodies in our 115 panel, all mAbs in groups A and B inhibited ACE2 binding to spike protein, mAbs in groups C 116 and D variably inhibited ACE2 binding, and mAbs in group E failed to inhibit ACE2 binding 117 (Fig 1) . 118

The mAbs also were tested for cross-reactive binding to the SARS-CoV-1 spike protein. 119

The majority of mAbs in group D, which competed with the cross-reactive mAb CR3022 for 120 spike binding, cross-reacted with SARS-CoV-1 spike protein, indicating they bind conserved 121 sarbecovirus epitopes. MAbs in groups A, B, and C did not bind to SARS-CoV-1 (Fig 1) , and 122 one group E mAb recognized SARS-CoV-1. Based on competition analysis, many anti-RBD 123 mAbs in our panel bind within or proximal to the RBM and are type-specific for SARS-CoV-2, 124 whereas those binding near the base of the RBD are more cross-reactive with SARS-CoV-1. 125

Anti-SARS-CoV-2 RBD mAbs neutralize SARS-CoV-2 with varying potency. We 126 next determined the neutralizing activity of mAb hybridoma supernatants using a focus-127 reduction neutralization test (FRNT) and Vero E6 cells (Case et al., 2020) with the WA1/2020 128 SARS-CoV-2 strain. Antibody concentrations were quantified by ELISA and used to calculate 129 half-maximal inhibitory concentrations (EC50 values). The most potently inhibitory mAbs 130 activity (EC50: 20 -100 ng/mL), although the majority were weakly inhibitory. Group E mAbs 133 were weakly neutralizing and did not block ACE2 binding. 134 A subset of mAbs from groups A, B, C, and D were selected for detailed study. We chose 135 two mAbs with the highest neutralization potency from each group; in cases where mAbs had 136 high variable region sequence similarity, we selected only one of these mAbs for further study. 137

We also selected SARS2-03, as it was one of the few neutralizing mAbs that did not block ACE2 138

binding. Nine mAbs were purified and retested for neutralization potency by FRNT using Vero 139 cells and the WA1/2020 isolate (Fig 2A-B) . Again, the most potently neutralizing purified mAbs 140 belonged to groups A, B, and C, with less inhibitory activity in those from group D. We also 141 characterized these nine mAbs for competition binding with each other (Fig S1) . The two group 142 A mAbs (SARS2-34 and SARS2-71) competed for spike binding only with each other. In 143 contrast, mAbs in groups B (SARS2-02 and SARS2-55) and C (SARS2-01 and SARS2-38) 144 competed for spike binding across both groups. SARS2-03, a group D mAb, did not bind spike 145 efficiently in the presence of group B or C mAbs and blocked binding of group C mAb SARS2-146 01. SARS2-10 and SARS2-31, the other two group D mAbs, however, competed only with each 147 other. Together, these results suggest that mAbs in group C may have overlapping epitopes with 148 group B mAbs and group D mAb SARS2-03, whereas group A mAbs and the remaining group D 149 mAbs likely engage physically distinct epitopes. 150

We 151 investigated whether the anti-SARS-CoV-2 mAbs inhibited infection at a pre-or post-attachment 152 step of the entry process. For these experiments, we selected one representative mAb from 153 groups A, B, and C (SARS2-34, SARS2-02, and SARS2-38, respectively) and two mAbs from 154 group D (SARS2-10 and SARS2-03, which respectively blocks or does not block ACE2 155 J o u r n a l P r e -p r o o f binding). We compared the neutralization potency of mAbs when added before or after virus 156 absorption to Vero E6 cells. All mAbs retained neutralizing activity when added post-157 attachment, although the potency of groups A, B, and C mAbs SARS2-02, SARS2-34, and 158 SARS2-38 was reduced slightly (~2-to 4-fold, p < 0.05) relative to pre-attachment neutralization 159 titers (Fig 2C-D) . SARS2-10, a group D mAb, also showed a ~5-fold decrease (p < 0.0001) in 160 neutralizing activity when added after attachment. In contrast, SARS2-03, another group D mAb, 161 and the only mAb in this smaller panel that did not block ACE2-spike interactions, had similar 162 neutralization potencies (p = 0.79) when added before or after cell attachment. These data 163 suggest that mAbs that inhibit spike protein binding to ACE2 neutralize SARS-CoV-2 slightly 164 more efficiently when given at a pre-attachment step, although all of the mAbs tested retained 165 the ability to inhibit infection when given after virus attachment to cells. 166

To determine the impact of entry factor expression on target cells on virus neutralization, 167 we extended these findings to cells that ectopically express human ACE2 and TMPRSS2. In 168 contrast to the relatively minor change in neutralization potency seen with all mAbs for pre-169 versus post-attachment observed using Vero E6 cells, mAbs no longer efficiently neutralized 170 SARS-CoV-2 infection when added after attachment to Vero-TMPRSS2-ACE2 cells, although 171 pre-attachment neutralization activity remained intact (Fig 2E) . Thus, the ability of anti-SARS-172

CoV-2 mAbs to neutralize at a post-attachment step was modulated by expression levels of viral 173 entry factors and the cell line. 174

We also tested the ability of the mAbs to block directly virus attachment to cells, 175

including Vero E6, Vero-TMPRSS2, and Vero-TMPRSS2-ACE2 cells. None of the mAbs 176 efficiently blocked SARS-CoV-2 attachment to Vero or Vero-TMPRSS2 cells (Fig 2F) . control with inhibition by only SARS2-38 attaining statistical significance (Fig 2G) . This result 183 suggests that the anti-RBD mAbs can inhibit viral attachment to cells, but this activity depends 184 on levels of human ACE2 expression. Since the mAbs did not efficiently inhibit attachment to 185 Vero E6 cells lacking human ACE2 expression, we tested whether they block a later step in the 186 entry process by using a virus internalization assay (Dejarnac et al., 2018; Earnest et al., 2021) . 187

In Vero E6 cells, pre-incubation with all of the anti-RBD mAbs tested resulted in reduced levels 188 of internalized virus (Fig 2H) . 189

Because we observed cell type-dependent differences in the mechanism of neutralization, 190

we tested the effect of cell substrate on the inhibitory potency of our anti-RBD mAbs by FRNT. 191 Notably, the anti-RBD mAbs neutralized SARS-CoV-2 WA1/2020 equivalently in Vero E6, 192 Vero-TMRPSS2, and Vero-TMPRSS2-ACE2 cells (Fig S2) . Animals treated with group D mAbs SARS2-10, SARS2-31 or SARS2-03 generally were less 224 protected against virus-induced weight loss (Fig 4D) . 225

To corroborate these findings, we measured the effect of mAb treatment on viral burden 226 in the nasal washes and lungs on 7 dpi. The greatest decreases in viral RNA levels (~30 to 100-227 fold) in the nasal washes relative to isotype control mAb-treated mice were observed in animals 228 treated with mAbs in groups B (SARS2-02 and SARS2-55) and C (SARS2-01 and SARS2-38) 229

( Fig 4E) . The largest reductions in viral RNA levels in the lung (~100 to 1,000-fold) again were 230 observed for mice treated with mAbs in groups B (SARS2-02 and SARS2-55) and C (SARS2-231 38) (Fig 4F) . A smaller (~10-fold) decrement of virus RNA levels in the lung was observed for 232 group D mAb SARS2-03. We also measured effects on infectious viral load in the lung by 233 plaque assay for a subset of representative mAbs from each group. Whereas group A mAb 234 SARS2-71 did not decrease the number of plaque-forming units (PFU) in the lung relative to the 235 isotype control mAb-treated mice, SARS2-02, SARS2-38, and SARS2-03 all reduced infectious 236 virus levels to the limit of detection of the assay (Fig 4G) . The lack of protection conferred by 237 SARS2-71 in vivo was unanticipated given its neutralizing activity in cell culture (EC50 of 8 238 ng/mL, Fig 2) . Sequencing of viral RNA from the lungs of SARS2-71-treated mice at 7 dpi 239 revealed an S477N mutation in all samples, which was not present in the input WA1/2020 virus. 240

Notably, S477N also emerged in vitro as an escape mutant under SARS2-71 selection pressure 241 using the VSV-eGFP-SARS-CoV-2-S virus (Fig 3A) . Thus, despite its potent inhibitory activity 242 in reduced cytokine and chemokine levels relative to isotype control mAb-treated mice, with 249 levels equivalent to those seen in naïve mice (Fig S3) . In contrast, treatment with SARS2-71 and 250 SARS2-03 did not result in these reductions. 251

To test for post-exposure therapeutic protection against SARS-CoV-2 challenge, we 252 cloned the variable regions of group B mAb SARS2-02 and group C mAb SARS2-38 and 253 inserted them into a human IgG1 backbone to make chimeric antibodies. We did this since 254 chimeric, humanized, or fully-human mAbs are more likely to be used in humans, and because 255

Fc effector functions contribute to the therapeutic activity of neutralizing SARS-CoV-2 mAbs in 256 vivo ; the original murine IgG1 isotype of these mAbs binds poorly to 257 activating murine FcγRI and FcγRIV, whereas human IgG1 binds these murine Fc receptors with 258 higher affinity and thus could have enhanced effector function (Dekkers et al., 2017) . We 259 confirmed the neutralizing activity of the chimeric mAbs hSARS2-02 and hSARS-38 relative to 260 the original murine versions of the mAbs (Fig S4A) . Next, we inoculated K18-hACE2 mice with 261 10 3 FFU of SARS-CoV-2 WA1/2020. Twenty-four hours later, we administered a single 200 µg 262 (10 mg/kg) dose of hSARS2-02, hSARS2-38, or an isotype control mAb. Both hSARS2-02 and 263 hSARS2-38 protected against weight loss following infection (Fig 4H) . At 7 dpi, hSARS2-38 264 reduced viral RNA levels in the lung and heart by ~10,000-fold, whereas hSARS2-02 reduced 265 infection by only ~10-100 fold in these tissues (Fig 4I) . These data demonstrate that mAb 266 neutralization potency in vitro does not directly predict protective efficacy in vivo. and showed no inhibitory activity against B.1.617.1, which encodes E484Q and L452R 282 mutations (Fig 5B and S4B ). SARS2-38 did not lose potency against any of the variant viruses, 283

with EC50 values ranging from 1-7 ng/mL across the panel tested (Fig 5C and S4B) . 284

To expand this analysis, we tested the VSV-eGFP-SARS-CoV-2-S viruses that were 285 resistant to SARS2-02 and SARS2-38 for neutralization using full dose response curves analysis. 286 SARS2-02 showed ~20-fold reduced potency against E484K, ~100-fold reduced potency against 287 L452R and G446V, and did not neutralize G446D at the highest concentration of mAb tested 288 (Fig S4C) . SARS2-38 showed virtually no neutralizing activity against K444E, K444N, G446D, 289 or G446V mutants even at the highest concentration (1 g/ml) of mAb tested (Fig S4C) . Despite 290 these results with VSV-eGFP-SARS-CoV-2-S viruses, when we serially passaged authentic 291 WA1/2020 D614G or Wash-B.1.351 SARS-CoV-2 in Vero-TMPRSS2-ACE2 cells in the 292 presence of neutralizing mAbs, we readily isolated resistant viruses following SARS-02 but not 293 SARS2-38 selection with both strains. 294

We tested SARS2-02 and SARS2-38 for protection against Wash-B.1.351 in K18-hACE2 295 mice as pre-exposure prophylaxis or post-exposure therapy. Animals treated with 100 µg of 296 SARS2-02 or SARS2-38 24 h prior to infection were protected from weight loss (Fig 5D) , 297 despite the reduced neutralization potency of SARS2-02 against Wash B.1.351. SARS2-38 298 treatment greatly reduced viral titers in the lung, nasal washes, heart, and brain at 7 dpi compared 299 to the isotype control-treated mice, whereas SARS2-02 had less protective effect (Fig 5E) . When 300 hSARS2-02 and hSARS2-38 were administered to the K18-hACE2 transgenic mice as therapy 301 24 h after infection with Wash-B.1.351, a similar phenotype was observed: while they both 302 protected mice against weight loss (Fig 5F) , hSARS2-38 resulted in a greater reduction in viral 303 titers at 7 dpi in the lung, heart, and brain than hSARS2-02 (Fig 5G) . We also tested hSARS2-38 304 for therapeutic protection against another variant, B.1.617.1, in K18-hACE2 mice. When 305 administered 24 h after infection with B.1.617.1, hSARS2-38 protected mice against weight loss 306 and viral infection in the lung, heart, and brain (Fig 5H-I) . To evaluate further its therapeutic 307 window, we administered hSARS2-38 two days after Wash-B.1.351 inoculation in K18-hACE2 308 mice. hSARS2-38 treatment fully protected mice against weight loss and lethality following 309

Wash-B.1.351 infection (Fig 5J-K) . Finally, hSARS2-38 also protected Syrian golden hamsters 310 from Wash-B.1.351 when administered 24 h prior to infection, with hSARS2-38-treated 311 hamsters showing no weight loss and decreased viral RNA and infectious virus levels in the lung 312 (Fig S4D-F) . Thus, SARS2-38 neutralizes a large panel of SARS-CoV-2 circulating variants in 313 vitro and confers protection against multiple variants in vivo. 314

To 315 define further the mechanistic basis for the broad and potent neutralization by SARS2-38, we 316 first analyzed the interaction of antigen binding fragments (Fab) of SARS2-38 with SARS-CoV-317 2 spike using biolayer interferometry (BLI). SARS2-38 bound spike with high monovalent 318 affinity (kinetically derived K D of 6.5 nM) and had a half-life of 4.8 min (Fig S5A) . To 319 understand the basis for this binding structurally, we performed cryo-electron microscopy (cryo-320 EM) on complexes of SARS2-38 Fab and the SARS-CoV-2 spike protein (Fig S5B and Table  321 S1). Using a large spherical mask and an ab initio spike density reference, we generated three-322 dimensional classes to sample the oligomeric states of the Fab/spike complex, and the class of 323 highest resolution and clearest Fab density was refined further. This class consisted of trimeric 324 spike with all RBDs in the down position (D/D/D) and one RBD bound by Fab (Fig 6A and  325   S6A ). Using non-uniform refinement, we achieved an overall resolution of 3.20 Å, with local 326 resolution ranging from ~2.5 Å in the core of the spike to ~5.5 Å in the constant region of the 327 Fab, which was visible only at high contour (Fig S6B-D) . Other binding configurations also were 328 seen, although these classes aligned less clearly. This local refinement of the Fv/RBD complex achieved a nominal resolution of 3.16 Å, allowing 340 placement of the protein backbone, secondary structures, and most side chains at the interface 341 (Fig S5B and S6E-F) . The SARS2-38 Fv sits atop three loops protruding at the proximal end of 342 the RBM between helix α1 and strand β1 (contact residue T345), strands β4 and β5 (N439-G446, 343 N448-Y451), and strand β6 and helix α5 (P499-T500; Fig 6AB) ; these results correspond well 344 with our VSV-based escape mutant mapping (Fig 3) . All three light chain CDRs contact loop β4-345 β5, with CDR2 and CDR3 forming additional contacts with loops α1-β1 and β6-α5, respectively 346 ( Fig 6C) . In comparison, the heavy chain interacts in a more limited manner with loops β4-β5 347 and β6-α5 via CDR2 and CDR3 (Fig 6C) . CDR1 of the heavy chain makes no contact at all with 348 the RBD. The heavy chain does, however, engage ACE2 contact residues of the RBM (Fig 6C) . The SARS2-38 epitope is conserved among circulating SARS-CoV-2 variants of 353 concern. SARS2-38 potently neutralized all tested VOCs. To understand this broadly-354 neutralizing activity, we mapped the SARS2-38 epitope alongside VOC mutations within the 355 RBD (Fig 6D, left panel and Fig 6E) . One mutation in the SARS2-38 footprint, N439K, is 356 present in variant B.1.222 and resides at the periphery of the epitope. However, B.1.222 357 remained sensitive to neutralization by SARS2-38, and escape mutants at this residue were not 358 generated in vitro, suggesting that N439 is not critical for SARS2-38 binding. N439 forms no 359 close contacts (<3.9 Å) with SARS2-38 and accounts for only 1.4% of total buried surface area 360 at the interface ( Table S2) . The SARS2-38 epitope includes no other residues corresponding to 361 VOC mutations, which explains its performance against these variants. Notwithstanding this, we 362 could select escape mutations in vitro in the context of VSV-eGFP-SARS-CoV-2-S chimeric 363 virus, namely K444E/N and G446D/V substitutions, which reside on the β4-β5 loop central to 364 the SARS2-38 epitope (Fig 6D-E) We then developed a log-scale conservation score for RBD residues 333-520. In this model, 374 perfect conservation of the reference amino acid (from 2019n-CoV/WA1/2020) across all 375 isolates corresponds to a score of 1, and complete loss of the reference amino acid results in a 376 score of 0. Visualizing these scores on a color-coded RBD surface rendering (blue = 1, more 377 conserved; red = 0, more variable) revealed that the RBM is generally more variable than the rest 378 of the RBD, with VOCs clearly seen as red patches (Fig 6D, right panel) . This analysis also 379 suggested that in addition to not being affected by the VOCs tested in this study, SARS2-38 380 targets a portion of the RBM that is conserved among circulating SARS-CoV-2 variants. The 429, B.1.298, B.1.222, B.1.617.1, B.1.617.2, B.1.526+S477N, Expifectamine 293 (Thermo Fisher Scientific). Supernatants were harvested after 5-6 days, 774 purified by affinity chromatography (Protein A Sepharose, GE), and desalted with a PD-10 775 (Cytiva) column. 776

to quantify the binding capacity of SARS2-38 Fab fragments to trimerized SARS-CoV-2 spike. 778 10 µg/mL of biotinylated spike was immobilized onto streptavidin biosensors (ForteBio) for 3 779 min. After a 30 sec wash, the pins were submerged in running buffer (10 mM HEPES, 150 mM 780 NaCl, 3 mM EDTA, 0.05% P20 surfactant, and 1% BSA) containing SARS2-38 Fab ranging 781 from 1 to 1,000 nM, followed by a dissociation step in running buffer alone. The BLI signal was 782 recorded and analyzed using BIAevaluation Software (Biacore). incubated with mAd dilutions for 1 h at 4°C. Virus then was allowed to internalize for 30 min at 853 37°C, and subsequently cells were overlaid with methylcellulose as described above. Thirty 854 hours later, plates were fixed with 4% PFA and analyzed for antigen-specific foci as described 855 above for FRNTs. Due to less efficient binding of virus to cells at 4°C, a 2-fold higher amount of 856 input virus was used in the post-attachment assay; however, this is unlikely to affect mAb 857 potency, as the final FFU count following removal of unbound virus in the post-attachment assay 858 (which is prior to mAb incubation) is similar to that used in the pre-attachment assay. 859 Attachment inhibition assay. SARS-COV-2 was incubated with mAbs at 10 µg/mL for 860 1 h at 4°C. The mixture then was added to pre-chilled Vero E6, Vero-TMPRSS2, Vero-861 TMPRSS2-ACE2, or Calu-3 cells at an MOI of 0.005 and incubated at 4°C for 1 h. Cells were 862 washed six times with chilled PBS before addition of lysis buffer and extraction of RNA using 863

MagMax viral RNA isolation kit (Thermo Fisher Scientific) and a Kingfisher Flex 96-well 864 extraction machine (Thermo Fisher Scientific). SARS-CoV-2 RNA was quantified by qRT-PCR 865 using the N-specific primer/probe set described below. GAPDH was measured using a 866 predesigned primer/probe set (IDT PrimeTime Assay Hs.PT.39a.22214836). Viral RNA levels 867 were normalized to GAPDH, and the fold change was compared with isotype control mAb. For 868 each cell type, a control with a 4-fold lower MOI (0.00125) was included to demonstrate 869 detection of decreased viral RNA levels. 870

Virus internalization assay. SARS-COV-2 was incubated with mAbs at 10 µg/mL for 1 871 h at 4°C. The mixture was then added to pre-chilled Vero E6 cells at an MOI of 0.005 and 872 incubated at 4°C for 1 h. Cells were washed twice with chilled PBS to remove unbound virus, 873 and subsequently incubated in DMEM at 37°C for 30 min to allow virus internalization. Cells 874 then were treated with proteinase K and RNaseA at 37°C for 10 min to removed uninternalized 875

virus. Viral and cellular RNA were extracted and analyzed as described above for the attachment 876 inhibition assay. A no internalization control was included, where proteinase K and RNase A 877 treatments were performed directly after washing, without an internalization step. 878

Measurement of viral burden and cytokine and chemokine levels. On 7 dpi, mice 879 were euthanized and organs were collected. Nasal washes were collected in 0.5 mL of PBS. 880

Organs were weighed and homogenized using a MagNA Lyser (Roche). Viral RNA from 881 homogenized organs or nasal wash was isolated using the MagMAX Viral RNA Isolation Kit 882 (ThermoFisher) and measured by TaqMan one-step quantitative reverse-transcription PCR (RT-883 qPCR) on an ABI 7500 Fast Instrument. Viral burden is expressed on a log 10 scale as viral RNA 884 per mg for each organ or total nasal wash after comparison with a standard curve produced using 885 

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SARS-CoV-2 RBD antibodies that maximize 1101 breadth and resistance to escape

Complete map of SARS-1103

CoV-2 RBD mutations that escape the monoclonal antibody LY-CoV555 and its cocktail with 1104 LY-CoV016

Neutralizing and protective human 1107 monoclonal antibodies recognizing the N-terminal domain of the SARS-CoV-2 spike protein

Human monoclonal antibody 1111 combination against SARS coronavirus: synergy and coverage of escape mutants

Circulating SARS-CoV-2 spike 1115 N439K variants maintain fitness while evading antibody-mediated immunity

Ultrapotent human antibodies protect against 1119 SARS-CoV-2 challenge via multiple mechanisms

Structural basis for broad sarbecovirus 1123 neutralization by a human monoclonal antibody

Coronaviridae Study Group of the International Committee on Taxonomy of Viruses

The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and 1126 naming it SARS-CoV-2

Safety and efficacy of the ChAdOx1 1129 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised 1130 controlled trials in Brazil, South Africa, and the UK

SPHIRE-crYOLO is a fast and accurate fully automated 1134 particle picker for cryo-EM

Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7. 1137

REGN-COV2, a Neutralizing Antibody Cocktail, in 1140 Outpatients with Covid-19

Escape from neutralizing 1143 antibodies by SARS-CoV-2 spike protein variants

SARS-CoV-2 1146 501Y.V2 escapes neutralization by South African COVID-19 donor plasma

SARS-CoV-2 infection of human ACE2-transgenic mice 1150 causes severe lung inflammation and impaired function

Human neutralizing antibodies against SARS-CoV-2 1154 require intact Fc effector functions for optimal therapeutic protection

K18-hACE2 mice 1158 develop respiratory disease resembling severe COVID-19

1161 (2020). A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and 1162 SARS-CoV

Gctf: Real-time CTF determination and correction

MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron 1167 microscopy

New tools for automated high-resolution cryo-EM structure determination in 1170 RELION-3

A Bayesian approach to beam-induced 1172 motion correction in cryo-EM single-particle analysis

Potently neutralizing and protective human 1176 antibodies against SARS-CoV-2

Competition binding analysis. The assay was performed as described previously (Zost 730 et al., 2020) . Briefly, for screening study wells of 384-well microtiter plates were coated with 1 731 μg/mL of purified SARS-CoV-2 S6P ecto protein at 4 °C overnight. Plates were blocked with 2% 732 bovine serum albumin (BSA) in DPBS-T for 1 h. Mouse hybridoma culture supernatants were 733 diluted five-fold in blocking buffer, added to the wells (20 μl per well) in duplicates for each 734 tested reference mAb and incubated for 1 h at room temperature. Biotinylated reference human 735 mAbs with known epitope specificity (COV2-2130, COV2-2196 (Zost et al., 2020) , and CR3022 736 Neutralization assays. FRNTs were performed as described (Case et al., 2020) . Briefly, 833 serial dilutions of antibody were incubated with 2 x 10 2 FFU of SARS-CoV-2 for 1 h at 37°C. 834Immune complexes were added to cell monolayers (Vero E6 cells or other cell lines where 835 indicated) and incubated for 1 h at 37°C prior to the addition of 1% (w/v) methylcellulose in 836 MEM. Following incubation for 30 h at 37°C, cells were fixed with 4% paraformaldehyde 837 (PFA), permeabilized and stained for infection foci with SARS2-16 (hybridoma supernatant 838 diluted 1:6,000 to a final concentration of ~20 ng/mL) when using SARS-CoV-2 isolate 839 WA1/2020, or with a mixture of mAbs that bind various epitopes on the RBD and NTD of spike 840 (SARS2-02, SARS2-11, SARS2-31, SARS2-38, SARS2-57, and SARS2-71; diluted to 1 µg/mL 841 total mAb concentration) for the VOCs. Antibody-dose response curves were analyzed using 842